Columns sanitization

Table 3.6. The range of CIP agents often used to clean/sanitize chromatographic columns. Most CIP protocols would make use of two or more of these agents, allowing them to sequentially percolate through the column at a slow flow rate. Contact time can range from several minutes to overnight. NaOH is particularly effective at removing most contaminant types...

A wide variety of equipment is used for chromatography and, in general, such equipment should be dedicated to the purification of one product and should be sterilized or sanitized between batches. The use of the same equipment at different stages of processing should be discouraged. Acceptance criteria, lifespan and sanitation or sterilization method of columns should be defined. 

Chromatographic resins contain a great deal of surface area, and a clear understanding of what is occurring on the surface in terms of carryover of risk factors is not always possible. Cleaning and sanitization, as well as column lifetime, are issues that must be addressed in development and validated at pilot or full scale. 

The germ reduction of chromatographic resins is conventionally carried out by chemical treatments before or after packing a column. The choice of the sanitizing agent is largely dependent on the sensitivity of the resin. 

Sodium hydroxide is widely used in the biotech industry to both regenerate and sanitize chromatography resins to a certain extent. The practice of using sodium hydroxide as a cleaning agent varies a lot in terms of concentration (0.1 to 1 N), incubation times (hours to several days), temperature, and column volumes passed through the column (1 to more than 10). 

Jungbauer, A., Lettner, H. P., Guerrier, L., and Boschetti, E. (1974). Chemical sanitization in process chromatography. Part 2 In situ treatment of packed columns and long-term stability of resins. Bio-Pharm. 7, 37-42. 

Lapidus, L., and N.R. Amundson. 1952. Mathematics of adsorption in beds. VI. The effects of longitudinal diffusion in ion exchange and chromatographic columns. J, Phys. Chem. 56 984-988. Li, W.H. 1972a. Effects of dispersion on DO-SAG in uniform flow. J. Sanit. Eng. 98 169-182. 

Sanitary Design and Sanitization Sanitary design is necessary to ensure hygienic performance and thorough cleaning standard column attributes to minimize bacterial contamination are listed below ... 

Use 0.5 M NaOH for cleaning and sanitizing the fluid path and columns, except where it is incompatible with the matrix (e.g. porous glass) or ligand (e.g. Protein G, antibodies). Treatment for 30-60 min will remove most strong adsorbed molecules, destroy microbes and viruses, and remove endotoxin (6). Removal of the NaOH is easy to monitor and disposal is not a problem. 

This is not eliminated by a blank elution cycle because a trace of the first andbcxly may remain tightly boxmd only to be displaced when the column is loaded with Ig again. The problem can be overcome by a short wash with NaOH. Contrary to expectation, there is minimal damage to the Protein A matrix (10). However, this cleaning procedure is a veiy effective way of sanitizing the column and preventing the accumulation of bacterial endotoxin. 

Ozone. Ozone is a strong oxidizer formed by the excitation of molecular oxygen into atomic oxygen in an environment that allows it to recombine into O3. Ozone is used as an oxidant and supplemental sanitizer in both pools and especially in spas. The half-life of ozone is on the order of a few seconds (Haas, 1991). Because of the instability, it is not possible to maintain an ozone residual in the entire water column. Ozone must be used in conjunction with a primary sanitizer. Hypochlorous acid, hypobromous acid and PHMB are suitable as primary sanitizers for ozone system, although the consumption of PHMB will be increased by 25 to 50 percent (Unhoch and Vore, unpublished data). 

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